Novel specific hcv ns3 protease inhibitors

ABSTRACT

The present invention is directed to compounds, compositions and methods for treating or preventing viral infections, in particular, HCV in human patients or other animal hosts.

FIELD OF THE INVENTION

The present invention is directed to compounds methods and compositionsthat are useful as inhibitors of the hepatitis C virus (HCV) NS3protease, the synthesis of such compounds, and the use of such compoundsfor treating HCV infection and or reducing the likelihood or severity ofsymptoms of HCV infection.

This application claims priority to U.S. Provisional Application No.61/408,989, filed on Nov. 1, 2010, the contents of which are herebyincorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) has infected more than 180 million peopleworldwide. It is estimated that three to four million persons are newlyinfected each year, 70% of whom will develop chronic hepatitis. HCV isresponsible for 50-76% of all liver cancer cases, and two thirds of allliver transplants in the developed world. Standard therapy (pegylatedinterferon alpha plus ribavirin) is only effective in 50-60% ofpatients; however, its effectiveness is not well understood and it isassociated with significant side-effects. Therefore, there is an urgentneed for new drugs to treat and/or cure HCV (1: Chen K X, Njoroge F G. Areview of HCV protease inhibitors. Curr Opin Investig Drugs. 2009 8,821-37; 2: Garg G, Kar P. Management of HCV infection: current issuesand future options. Trop Gastroenterol. 2009 30, 11-8; 3: Pereira A A,Jacobson I M. New and experimental therapies for HCV. Nat RevGastroenterol Hepatol. 2009 7, 403-11).

The HCV genome comprises a positive-strand RNA enclosed in anucleocapsid and lipid envelope and consists of 9.6 kb ribonucleotides,which encodes a large polypeptide of about 3000 amino acids (Dymock etal. Antiviral Chemistry & Chemotherapy 2000, 11, 79). Followingmaturation, this polypeptide is cut into at least 10 proteins. The NS3serine protease, located in the N-terminal domain of the NS3 protein,mediates all of the subsequent cleavage events downstream in thepolyprotein. Because of its role, the NS3 serine protease is an idealdrug target and previous research has shown hexapeptides as well astripeptides show varying degrees of inhibition, as discussed in U.S.patent applications US2005/0020503, US2004/0229818, and US2004/00229776.Macrocyclic compounds that exhibit anti-HCV activity have also beendisclosed in International patent applications nos. W020061119061,W02007/015855 and W02007/016441 (all Merck & Co., Inc.).

The discovery of novel antiviral strategies to selectively inhibit HCVreplication has long been hindered by the lack of convenient cellculture models for the propagation of HCV. This hurdle has been overcomefirst with the establishment of the HCV replicon system in 1999(Bartenschlager, R., Nat. Rev. Drug Discov. 2002, 1, 911-916 andBartenschlager, R., J. Hepatol. 2005, 43, 210-216) and, in 2005, withthe development of robust HCV cell culture models (Wakita, T., et al.,Nat. Med. 2005, 11, 791-6; Zhong, J., et al., Proc. Natl. Acad. Sci.U.S.A. 2005, 102, 9294-9; Lindenbach, B. D., et al., Science 2005, 309,623-6).

It would be advantageous to provide new antiviral or chemotherapyagents, compositions including these agents, and methods of treatmentusing these agents, particularly to treat drug resistant or mutantviruses. The present invention provides such agents, compositions andmethods.

SUMMARY OF THE INVENTION

The present invention provides compounds, methods and compositions fortreating or preventing HCV infection in a host. The compounds have thefollowing general formula:

Where R, J, and J¹ are as defined hereinbelow.

The methods involve administering a therapeutically orprophylactically-effective amount of at least one compound as describedherein to treat or prevent an infection by, or an amount sufficient toreduce the biological activity of HCV infection. The pharmaceuticalcompositions include one or more of the compounds described herein, incombination with a pharmaceutically acceptable carrier or excipient, fortreating a host with HCV. The formulations can further include at leastone further therapeutic agent. In addition, the present inventionincludes processes for preparing such compounds.

Hepatitis C replicons require viral helicase, protease, and polymeraseto be functional in order for replication of the replicon to occur. Thereplicons can be used in high throughput assays, which evaluate whethera compound to be screened for activity inhibits the ability of HCVhelicase, protease, and/or polymerase to function, as evidenced by aninhibition of replication of the replicon.

DETAILED DESCRIPTION

The compounds described herein show inhibitory activity against HCV.Therefore, the compounds can be used to treat or prevent a viralinfection in a host, or reduce the biological activity of the virus. Thehost can be a mammal, and in particular, a human, infected with HCV. Themethods involve administering an effective amount of one or more of thecompounds described herein.

Pharmaceutical formulations including one or more compounds describedherein, in combination with a pharmaceutically acceptable carrier orexcipient, are also disclosed. In one embodiment, the formulationsinclude at least one compound described herein and at least one furthertherapeutic agent.

The present invention will be better understood with reference to thefollowing definitions:

I. Definitions

The terms “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

As used herein, the term “enantiomerically pure” refers to a compoundcomposition that comprises at least approximately 95%, and, preferably,approximately 97%, 98%, 99% or 100% of a single enantiomer of thatcompound.

As used herein, the term “substantially free of” or “substantially inthe absence or refers to a compound composition that includes at least85 to 90% by weight, preferably 95% to 98% by weight, and, even morepreferably, 99% to 100% by weight, of the designated enantiomer of thatcompound. In a preferred embodiment, the compounds described herein aresubstantially free of enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85 to 90% by weight, preferably 95% to 98% by weight,and, even more preferably, 99% to 100% by weight, of the compound, theremainder comprising other chemical species or enantiomers.

The term “alkyl,” as used herein, unless otherwise specified, refers to'a saturated straight, branched, or cyclic, primary, secondary, ortertiary hydrocarbons, including both substituted and unsubstitutedalkyl groups. The alkyl group can be optionally substituted with anymoiety that does not otherwise interfere with the reaction or thatprovides an improvement in the process, including but not limited to butlimited to halo, haloalkyl, hydroxyl, carboxyl, acyl, aryl, acyloxy,amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl,sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide,phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether,acid halide, anhydride, oxime, hydrazine, carbamate, phosphonic acid,phosphonate, either unprotected, or protected as necessary, as known tothose skilled in the art, for example, as taught in Greene, et al.,Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference. Specifically includedare CF₃ and CH₂CF₃

In the text, whenever the term C(alkyl range) is used, the termindependently includes each member of that class as if specifically andseparately set out. The term “alkyl” includes C₁₋₂₂ alkyl moieties, andthe term “lower alkyl” includes C₁₋₆ alkyl moieties. It is understood tothose of ordinary skill in the art that the relevant alkyl radical isnamed by replacing the suffix “-ane” with the suffix “-yl”.

The term “alkenyl” refers to an unsaturated, hydrocarbon radical, linearor branched, in so much as it contains one or more double bonds. Thealkenyl group disclosed herein can be optionally substituted with anymoiety that does not adversely affect the reaction process, includingbut not limited to but not limited to those described for substituentson alkyl moieties. Non-limiting examples of alkenyl groups includeethylene, methylethylene, isopropylidene, 1,2-ethane-diyl,1,1-ethane-diyl, 1,3-propane-diyl, 1,2-propane-diyl, 1,3-butane-diyl,and 1,4-butane-diyl.

The term “alkynyl” refers to an unsaturated, acyclic hydrocarbonradical, linear or branched, in so much as it contains one or moretriple bonds. The alkynyl group can be optionally substituted with anymoiety that does not adversely affect the reaction process, includingbut not limited to those described above for alkyl moeities.Non-limiting examples of suitable alkynyl groups include ethynyl,propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl,pentyn-2-yl, 4-methoxypentyn-2-yl, 3-methylbutyn-1-yl, hexyn-1-yl,hexyn-2-yl, and hexyn-3-yl, 3,3-dimethylbutyn-1-yl radicals.

The term “alkylamino” or “arylamino” refers to an amino group that hasone or two alkyl or aryl substituents, respectively.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, sulfur, nitrogen, or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis, and are described, for example,in Greene et al., Protective Groups in Organic Synthesis, supra.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one, two or three rings wherein such rings can beattached together in a pendent manner or can be fused. Non-limitingexamples of aryl include phenyl, biphenyl, or naphthyl, or otheraromatic groups that remain after the removal of a hydrogen from anaromatic ring. The term aryl includes both substituted and unsubstitutedmoieties. The aryl group can be optionally substituted with any moietythat does not adversely affect the process described herein forpreparing the compounds, including but not limited to but not limited tothose described above for alkyl moieties. Non-limiting examples ofsubstituted aryl include heteroarylamino, N-aryl-N-alkylamino,N-heteroarylamino-N-alkylamino, heteroaralkoxy, arylamino, aralkylamino,arylthio, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl,monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, heteroarylthio,heteroarylsulfinyl, heteroarylsulfonyl, aroyl, heteroaroyl, aralkanoyl,heteroaralkanoyl, hydroxyaralkyl, hydoxyheteroaralkyl, haloalkoxyalkyl,aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl,partially saturated heterocyclyl, heteroaryl, heteroaryloxy,heteroaryloxyalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, andheteroarylalkenyl, carboaralkoxy.

The terms “alkaryl” or “alkylaryl” refer to an alkyl group with an arylsubstituent. The terms “aralkyl” or “arylalkyl” refer to an aryl groupwith an alkyl substituent.

The term “halo,” as used herein, includes chloro, bromo, iodo andfluoro.

The term “acyl” refers to a carboxylic acid ester in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including but notlimited to methoxymethyl, aralkyl including but not limited to benzyl,aryloxyalkyl such as phenoxymethyl, aryl including but not limited tophenyl optionally substituted with halogen (F, Cl, Br, I), alkyl(including but not limited to C₁, C₂, C₃, and C₄) or alkoxy (includingbut not limited to C₁, C₂, C₃, and C₄), sulfonate esters such as alkylor aralkyl sulphonyl including but not limited to methanesulfonyl, themono, di or triphosphate ester, trityl or monomethoxytrityl, substitutedbenzyl, trialkylsilyl (e.g., dimethyl-t-butylsilyl) ordiphenylmethylsilyl. Aryl groups in the esters optimally comprise aphenyl group. The term “lower acyl” refers to an acyl group in which thenon-carbonyl moiety is lower alkyl.

The terms “alkoxy” and “alkoxyalkyl” embrace linear or branchedoxy-containing radicals having alkyl moieties, such as methoxy radical.The term “alkoxyalkyl” also embraces alkyl radicals having one or morealkoxy radicals attached to the alkyl radical, that is, to formmonoalkoxyalkyl and dialkoxyalkyl radicals. The “alkoxy” radicals can befurther substituted with one or more halo atoms, such as fluoro, chloroor bromo, to provide “haloalkoxy” radicals. Examples of such radicalsinclude fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy,trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, andfluoropropoxy.

The term “alkylamino” denotes “monoalkylamino” and “dialkylamino”containing one or two alkyl radicals, respectively, attached to an aminoradical. The terms arylamino denotes “monoarylamino” and “diarylamino”containing one or two aryl radicals, respectively, attached to an aminoradical. The term “aralkylamino”, embraces aralkyl radicals attached toan amino radical. The term aralkylamino denotes “monoaralkylamino” and“diaralkylamino” containing one or two aralkyl radicals, respectively,attached to an amino radical. The term aralkylamino further denotes“monoaralkyl monoalkylamino” containing one aralkyl radical and onealkyl radical attached to an amino radical.

The term “heteroatom,” as used herein, refers to oxygen, sulfur,nitrogen and phosphorus.

The terms “heteroaryl” or “heteroaromatic,” as used herein, refer to anaromatic that includes at least one sulfur, oxygen, nitrogen orphosphorus in the aromatic ring or a combination of two or moreheteroatoms (O, S, N, P) in an aromatic system. Both five membered andsix membered ring heteroaryls are contemplated herein, as are five andsix membered ring heteroaryls linked to a benzene ring, such asbenzofuran, benzthiophene, benzopyrrole, and the like.

The term “heterocyclic,” “heterocyclyl,” and cycloheteroalkyl refer to anonaromatic cyclic group wherein there is at least one heteroatom, suchas oxygen, sulfur, nitrogen, or phosphorus in the ring.

Nonlimiting examples of heteroaryl and heterocyclic groups includefuryl, furanyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl,tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl,isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl,isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl,isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, quinazolinyl,cinnolinyl, phthalazinyl, xanthinyl, hypoxanthinyl, thiophene, furan,pyrrole, isopyrrole, pyrazole, imidazole, 1,2,3-triazole,1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, pyrimidine orpyridazine, and pteridinyl, aziridines, thiazole, isothiazole,1,2,3-oxadiazole, thiazine, pyridine, pyrazine, piperazine, pyrrolidine,oxaziranes, phenazine, phenothiazine, morpholinyl, pyrazolyl,pyridazinyl, pyrazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl,pteridinyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl,imidazolopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine,N⁶-alkylpurines, N⁶-benzylpurine, N⁶-halopurine, N⁶-vinypurine,N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkyl purine,N⁶-thioalkyl purine, thymine, cytosine, 6-azapyrimidine,2-mercaptopyrmidine, uracil, N⁵-alkylpyrimidines, N⁵-benzylpyrimidines,N⁵-halopyrimidines, N⁵-vinylpyrimidine, N⁵-acetylenic pyrimidine,N⁵-acyl pyrimidine, N⁵-hydroxyalkyl purine, and N⁶-thioalkyl purine, andisoxazolyl. The heteroaromatic group can be optionally substituted asdescribed above for aryl. The heterocyclic or heteroaromatic group canbe optionally substituted with one or more substituent selected fromhalogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives, amido,amino, alkylamino, dialkylamino. The heteroaromatic can be partially ortotally hydrogenated as desired. As a nonlimiting example,dihydropyridine can be used in place of pyridine. Functional oxygen andnitrogen groups on the heterocyclic or heteroaryl group can be protectedas necessary or desired. Suitable protecting groups are well known tothose skilled in the art, and include trimethylsilyl,dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl,trityl or substituted trityl, alkyl groups, acyl groups such as acetyland propionyl, methanesulfonyl, and p-toluenelsulfonyl. The heterocyclicor heteroaromatic group can be substituted with any moiety that does notadversely affect the reaction, including but not limited to but notlimited to those described above for aryl.

The term “host,” as used herein, refers to a unicellular ormulticellular organism in which the virus can replicate, including butnot limited to cell lines and animals, and, preferably, humans.Alternatively, the host can be carrying a part of the viral genome,whose replication or function can be altered by the compounds of thepresent invention. The term host specifically refers to infected cells,cells transfected with all or part of the viral genome and animals, inparticular, primates (including but not limited to chimpanzees) andhumans. In most animal applications of the present invention, the hostis a human patient. Veterinary applications, in certain indications,however, are clearly contemplated by the present invention (such as foruse in treating chimpanzees).

The term “peptide” refers to various natural or synthetic compoundscontaining two to one hundred amino acids linked by the carboxyl groupof one amino acid to the amino group of another.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform of a compound that upon administration to a patient, provides theparent compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on functionalmoieties of the active compound. Prodrugs include compounds that can beoxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated,hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated,phosphorylated, or dephosphorylated to produce the active compound. Theprodrug forms of the compounds of this invention can possess antiviralactivity, can be metabolized to form a compound that exhibits suchactivity, or both.

II. Active Compound

The compounds described herein have the following general formula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein

J and J¹ can be present or absent, and when present, are independentlyselected from lower alkyl (C₁-C₆), aryl, arylalkyl, alkoxy, aryloxy,heterocyclyl, heterocyclyloxy, keto, hydroxy, amino, arylamino,carboxyalkyl, carboxamidoalkyl, halo, cyano, formyl, sulfonyl, orsulfonamido; and

R is C₁-C₁₀ alkyl, C₃₋₈ cycloalkyl, aryl, heteroaryl, or heterocyclyl.

III. Stereoisomerism and Polymorphism

The compounds described herein may have asymmetric centers and occur asracemates, racemic mixtures, individual diastereomers or enantiomers,with all isomeric forms being included in the present invention.Compounds of the present invention having a chiral center can exist inand be isolated in optically active and racemic forms. Some compoundscan exhibit polymorphism. The present invention encompasses racemic,optically active, polymorphic, or stereoisomeric forms, or mixturesthereof, of a compound of the invention, which possess the usefulproperties described herein. The optically active forms can be preparedby, for example, resolution of the racemic form by recrystallizationtechniques, by synthesis from optically active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase or by enzymatic resolution.

Optically active forms of the compounds can be prepared using any methodknown in the art, including but not limited to by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase.

Examples of methods to obtain optically active materials include atleast the following.

-   -   i) physical separation of crystals: a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization: a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions: a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis: a synthetic technique        whereby at least one step of the synthesis uses an enzymatic        reaction to obtain an enantiomerically pure or enriched        synthetic precursor of the desired enantiomer;    -   v) chemical asymmetric synthesis: a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which can be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations: a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations: a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions: this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors: a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography: a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase (including but not limited to via chiral HPLC). The        stationary phase can be made of chiral material or the mobile        phase can contain an additional chiral material to provoke the        differing interactions;    -   xi) chiral gas chromatography: a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents: a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes: a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane that allows only one        enantiomer of the racemate to pass through.

Chiral chromatography, including but not limited to simulated moving bedchromatography, is used in one embodiment. A wide variety of chiralstationary phases are commercially available.

IV. Compound Salt or Prodrug Formulations

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate.Suitable inorganic salts can also be formed, including but not limitedto, sulfate, nitrate, bicarbonate and carbonate salts.

Pharmaceutically acceptable salts can be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid, affording aphysiologically acceptable anion. Alkali metal (e.g., sodium, potassiumor lithium) or alkaline earth metal (e.g., calcium, magnesium) salts ofcarboxylic acids can also be made.

V. Methods of Treatment

Hosts, including but not limited to humans, infected with HCV or a genefragment thereof, can be treated by administering to the patient aneffective amount of the active compound or a pharmaceutically acceptableprodrug or salt thereof in the presence of a pharmaceutically acceptablecarrier or diluent. The active materials can be administered by anyappropriate route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid or solid form.

VI. Combination or Alternation Therapy

In one embodiment, the compounds of the invention can be employedtogether with at least one other antiviral agent.

Table of anti-HCV Compounds Approved or in Preclinical and ClinicalDevelopment Pharmaceutical Drug Name Drug Category Company PEGASYS Longacting interferon Roche pegylated interferon alfa-2a INFERGENInterferon, InterMune interferon alfacon-1 Long acting interferonOMNIFERON Interferon, Viragen natural interferon Long acting interferonALBUFERON Longer acting Human Genome interferon Sciences REBIFInterferon Ares-Serono interferon beta-1a Omega Interferon InterferonBioMedicine Oral Interferon Oral Interferon Amarillo Biosciences alphaInterferon Anti-fibrotic InterMune gamma-1b IP-501 Anti-fibroticInterneuron Merimebodib IMPDH inhibitor Vertex VX-497 (inosinemonophosphate dehydrogenase) AMANTADINE Broad Antiviral Agent Endo Labs(Symmetrel) Solvay IDN-6556 Apotosis regulation Idun Pharma. XTL-002Monclonal Antibody XTL HCV/MF59 Vaccine Chiron CIVACIR PolyclonalAntibody NABI Therapeutic vaccine Innogenetics VIRAMIDINE NucleosideAnalogue ICN ZADAXIN Immunomodulator Sci Clone (thymosin alfa-1) CEPLENEImmunomodulator Maxim histamine dihydrochloride VX 950/ ProteaseInhibitor Vertex/Eli Lilly LY 570310 ISIS 14803 Antisense IsisPharmaceutical/ Elan IDN-6556 Caspase inhibitor Idun Pharmaceuticals,Inc. http://www.idun.com JTK 003 Polymerase Inhibitor AKROS PharmaTarvacin Anti-Phospholipid Peregrine Therapy HCV-796 PolymeraseInhibitor ViroPharma/Wye CH-6 Serine Protease Schering ANA971Isatoribine ANADYS ANA245 Isatoribine ANADYS CPG 10101 ImmunomodulatorColey (Actilon) Rituximab Anti-CD20 Monoclonal Genetech/IDEC (Rituxam)Antibody NM283 Polymerase Inhibitor Idenix (Valopicitabine)Pharmaceuticals HepX ™-C Monclonal Antibody XTL IC41 Therapeutic VaccineIntercell Medusa Interferon Longer acting interferon Flamel TechnologiesE-1 Therapeutic Vaccine Innogenetics Multiferon Long Acting InterferonViragen BILN 2061 Serine Protease Boehringer - Ingelheim Interferonbeta-1a Interferon Ares-Serono (REBIF)

VII. Pharmaceutical Compositions

Hosts, including but not limited to humans, infected with hepatitis Cvirus (“HCV”), or a gene fragment thereof, can be treated byadministering to the patient an effective amount of the active compoundor a pharmaceutically acceptable prodrug or salt thereof in the presenceof a pharmaceutically acceptable carrier or diluent. The activematerials can be administered by any appropriate route, for example,orally, parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid or solid form.

A preferred dose of the compound will be in the range of between about0.1 and about 100 mg/kg, more generally, between about 1 and 50 mg/kg,and, preferably, between about 1 and about 20 mg/kg, of body weight ofthe recipient per day. The effective dosage range of thepharmaceutically acceptable salts and prodrugs can be calculated basedon the weight of the parent compound to be delivered. If the salt orprodrug exhibits activity in itself, the effective dosage can beestimated as above using the weight of the salt or prodrug, or by othermeans known to those skilled in the art.

The compound is conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3,000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1,000 mg is usually convenient.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound from about 0.2 to 70 μM,preferably about 1.0 to 15 μM. This can be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient can be administered at once, or canbe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, unitdosage forms can contain various other materials that modify thephysical form of the dosage unit, for example, coatings of sugar,shellac, or other enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup can contain,in addition to the active compound(s), sucrose or sweetener as asweetening agent and certain preservatives, dyes and colorings andflavors.

The compound or a pharmaceutically acceptable prodrug or salts thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, antifungals, anti-inflammatories or otherantivirals, including but not limited to nucleoside compounds. Solutionsor suspensions used for parenteral, intradermal, subcutaneous, ortopical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid; buffers, suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity, such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including but notlimited to implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters and polylactic acid. For example, enterically coatedcompounds can be used to protect cleavage by stomach acid. Methods forpreparation of such formulations will be apparent to those skilled inthe art. Suitable materials can also be obtained commercially.

Liposomal suspensions (including but not limited to liposomes targetedto infected cells with monoclonal antibodies to viral antigens) are alsopreferred as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art, for example, asdescribed in U.S. Pat. No. 4,522,811 (incorporated by reference). Forexample, liposome formulations can be prepared by dissolving appropriatelipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoylphosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol)in an inorganic solvent that is then evaporated, leaving behind a thinfilm of dried lipid on the surface of the container. An aqueous solutionof the active compound is then introduced into the container. Thecontainer is then swirled by hand to free lipid material from the sidesof the container and to disperse lipid aggregates, thereby forming theliposomal suspension.

The terms used in describing the invention are commonly used and knownto those skilled in the art. As used herein, the following abbreviationshave the indicated meanings:

-   aq aqueous-   CDI carbonyldiimidazole-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   EDC 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride-   EtOAc ethyl acetate-   h hour/hours-   HOBt N-hydroxybenzotriazole-   M molar-   min minute-   rt or RT room temperature-   TBAT tetrabutylammonium triphenyldifluorosilicate-   TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   THF tetrahydrofuran

IX. General Procedures for Preparing Active Compounds

Methods for the preparation of the compounds of this invention can beprepared as described in detail below in the “Specific Example” section,or by other methods known to those skilled in the art. It will beunderstood by one of ordinary skill in the art that these schemes are inno way limiting and that variations of detail can be made withoutdeparting from the spirit and scope of the present invention.

SPECIFIC EXAMPLES

Specific compounds which are representative of this invention wereprepared as per the following examples and reaction sequences; theexamples and the diagrams depicting the reaction sequences are offeredby way of illustration, to aid in the understanding of the invention andshould not be construed to limit in any way the invention set forth inthe claims which follow thereafter. The present compounds can also beused as intermediates in subsequent examples to produce additionalcompounds of the present invention. No attempt has necessarily been madeto optimize the yields obtained in any of the reactions. One skilled inthe art would know how to increase such yields through routinevariations in reaction times, temperatures, solvents and/or reagents.

Reagents and solvents were obtained from commercial suppliers and wereused without further purification. Methylene chloride was dried anddistilled over CaCl₂ and stored over molecular sieves 4 Å under argon.Tetrahydrofuran was dried over sodium/benzophenone ketyl under argon anddistilled prior to use. Flash chromatography purifications wereperformed on Macherey Nalgel silica gel (40-63 μM) as the stationaryphase or were conducted using packed RediSep® columns on a Teledyne IscoCombiflash® Companion® apparatus. Analytical High Performance LiquidChromatography-mass analysis (HPLC-MS):

HPLC-MS conditions A1: HPLS-MS were performed on a Waters Alliance 2790apparatus equipped with Photodiode Array Detector Waters 996 and aWaters Micromass Q-Tof using a BDS Hypersil 50×2.1, 3 μm. Elutingconditions comprised a linear gradient: 0% to 80% of MeCN/H₂O in 20minutes (containing 0.1% TFA in positive mode and without TFA innegative mode), flow rate 0.2 mL/min.

HPLC-MS conditions A2: HPLS-MS were performed on a Waters Alliance 2790apparatus equipped with Photodiode Array Detector Waters 996 and aWaters Micromass Q-Tof using a Nucleodur C18 Pyramid 50×2.1, 3 μm.Eluting conditions comprised a linear gradient: 0% to 80% of MeCN/H₂O in20 minutes (containing 0.1% TFA in positive mode and without TFA innegative mode), flow rate 0.3 mL/min.

HPLC-MS conditions B: HPLS-MS were performed on a Agilent HP-1100apparatus equipped with Photodiode Array Detector Agilent G1315A, apolymerlabs ELS2100 (DEDL) detector and a Agilent Simple Quad ESI formass analysis using a Agilent Zorbax XDB-C18 RP C18 45×4.6, 3.5 μm.Eluting conditions comprised a linear gradient: 10% to 100% in 4.5minutes of MeCN/H₂O (containing 0.05% TFA), flow rate 1.5 mL/min.

Low resolution mass spectra (MS) were obtained from an AppliedBiosystems SCIEX 3200 QTRAP in Atmospheric Pressure Ionization condition(API) in positive (ES+) or negative (ES−) mode.

High Resolution Mass Spectroscopy (HRMS) was obtained from a PerkinElmer apparatus.

NMR spectra were recorded on Bruker Avance 250 at 250 MHz for ¹H and 63MHz for ¹³C NMR in deuterared solvents and are referenced in ppmrelative to the solvent residual peak (see reference Gottlieb H. E. etal. J. Org Chem 1997 (2) 7512-7515).

Example 1 Synthesis of P2 Hydroxyproline Derivative

Example 2 Synthesis of 10

Analogs of Compound 10, in which J and J¹ are present, can be prepared,for example, by using a substituted form of the starting material:

where J and J¹ are as defined herein, or, where these moieties wouldinterfere with the coupling chemistry described in Scheme I, areprotected groups that can be converted to the desired J and J¹ moietiesafter the coupling chemistry is completed, or at a later step in theoverall synthesis.

Compounds of this formula are known, and can be prepared using no morethan routine experimentation. Those skilled in the art will readilyunderstand that incorporation of substituents onto the aryl ring can bereadily realized, either before the core structures are prepared, orafterward (i.e., the substituents can be present during key couplingsteps, or can be added after the unsubstituted compound (i.e., withoutthe J and/or J¹ moieties) has been prepared. Such substituents canprovide useful properties in and of themselves, or serve as a handle forfurther synthetic elaboration. One proviso is that such substitutionshould either survive the synthesis conditions, or should be added afterthe synthesis is otherwise complete.

For example, the aryl ring can be halogenated using various knownprocedures, which vary depending on the particular halogen. Examples ofsuitable reagents include bromine/water in concentrated HBr, thionylchloride, pyr-ICl, fluorine and Amberlyst-A. A number of other analogs,bearing substituents in a diazotized position of an aryl ring, can besynthesized from the corresponding aniline compounds, via the diazoniumsalt intermediate. The diazonium salt intermediates can be preparedusing known chemistry, for example, treatment of aromatic amines such asaniline with sodium nitrite in the presence of a mineral acid.

Diazonium salts can be formed from anilines, which in turn can beprepared from nitrobenzenes (and analogous amine-substituted heteroarylrings can be prepared from nitro-substituted heteroaryl rings). Thenitro derivatives can be reduced to the amine compound by reaction witha nitrite salt, typically in the presence of an acid. Other substitutedanalogs can be produced from diazonium salt intermediates, including,but are not limited to, hydroxy, alkoxy, fluoro, chloro, iodo, cyano,and mercapto, using general techniques known to those of skill in theart. Likewise, alkoxy analogues can be made by reacting the diazoniumsalt with alcohols. The diazonium salt can also be used to synthesizecyano or halo compounds, as will be known to those skilled in the art.Mercapto substitutions can be obtained using techniques described inHoffman et al., J. Med. Chem. 36: 953 (1993). The mercaptan so generatedcan, in turn, be converted to an alkylthio substitutuent by reactionwith sodium hydride and an appropriate alkyl bromide. Subsequentoxidation would then provide a sulfone. Acylamido analogs of theaforementioned compounds can be prepared by reacting the correspondingamino compounds with an appropriate acid anhydride or acid chlorideusing techniques known to those skilled in the art of organic synthesis.

Hydroxy-substituted analogs can be used to prepare correspondingalkanoyloxy-substituted compounds by reaction with the appropriate acid,acid chloride, or acid anhydride. Likewise, the hydroxy compounds areprecursors of both the aryloxy and heteroaryloxy via nucleophilicaromatic substitution at electron deficient aromatic rings. Suchchemistry is well known to those skilled in the art of organicsynthesis. Ether derivatives can also be prepared from the hydroxycompounds by alkylation with alkyl halides and a suitable base or viaMitsunobu chemistry, in which a trialkyl- or triarylphosphine anddiethyl azodicarboxylate are typically used. See Hughes, Org. React.(N.Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996)for typical Mitsunobu conditions.

Cyano-substituted analogs can be hydrolyzed to afford the correspondingcarboxamido-substituted compounds. Further hydrolysis results information of the corresponding carboxylic acid-substituted analogs.Reduction of the cyano-substituted analogs with lithium aluminum hydrideyields the corresponding aminomethyl analogs. Acyl-substituted analogscan be prepared from corresponding carboxylic acid-substituted analogsby reaction with an appropriate alkyllithium using techniques known tothose skilled in the art of organic synthesis.

Carboxylic acid-substituted analogs can be converted to thecorresponding esters by reaction with an appropriate alcohol and acidcatalyst. Compounds with an ester group can be reduced with sodiumborohydride or lithium aluminum hydride to produce the correspondinghydroxymethyl-substituted analogs. These analogs in turn can beconverted to compounds bearing an ether moiety by reaction with sodiumhydride and an appropriate alkyl halide, using conventional techniques.Alternatively, the hydroxymethyl-substituted analogs can be reacted withtosyl chloride to provide the corresponding tosyloxymethyl analogs,which can be converted to the corresponding alkylaminoacyl analogs bysequential treatment with thionyl chloride and an appropriatealkylamine. Certain of these amides are known to readily undergonucleophilic acyl substitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- orN-arylcarbamoyloxy-substituted compounds by reaction with N-alkyl- orN-arylisocyanates. Amino-substituted analogs can be used to preparealkoxycarboxamido-substituted compounds and urea derivatives by reactionwith alkyl chloroformate esters and N-alkyl- or N-arylisocyanates,respectively, using techniques known to those skilled in the art oforganic synthesis.

Similarly, benzene rings can be substituted using known chemistry,including the reactions discussed above. For example, the nitro group onnitrobenzene can be reacted with sodium nitrite to form the diazoniumsalt, and the diazonium salt manipulated as discussed above to form thevarious substituents on a benzene ring.

The substituents described above can therefore be added to the startingbenzene ring, and incorporated into the final compounds describedherein.

Example 3

Mitochondrial Toxicity Assays in HepG2 Cells:

i) Effect of compounds on Cell Growth and Lactic Acid Production: Theeffect on the growth of HepG2 cells was determined by incubating cellsin the presence of 0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM drug. Cells(5×10⁴ per well) were plated into 12-well cell culture clusters inminimum essential medium with nonessential amino acids supplemented with10% fetal bovine serum, 1% sodium pyruvate, and 1%penicillin/streptomycin and incubated for 4 days at 37° C. At the end ofthe incubation period the cell number was determined using ahemocytometer. Also taught by Pan-Zhou X-R, Cui L, Zhou X-J, SommadossiJ-P, Darley-Usmer V M. “Differential effects of antiretroviralnucleoside analogs on mitochondrial function in HepG2 cells”Antimicrob.Agents Chemother. 2000; 44: 496-503. To measure the effects of compoundson lactic acid production, HepG2 cells from a stock culture were dilutedand plated in 12-well culture plates at 2.5×10⁴ cells per well. Variousconcentrations (0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM) of test compoundwere added, and the cultures were incubated at 37° C. in a humidified 5%CO₂ atmosphere for 4 days. At day 4 the number of cells in each well wasdetermined and the culture medium collected. The culture medium wasfiltered, and the lactic acid content in the medium determined using acolorimetric lactic acid assay (Sigma-Aldrich). Since lactic acidproduct can be considered a marker for impaired mitochondrial function,elevated levels of lactic acid production detected in cells grown in thepresence of test compound would indicate a drug-induced cytotoxiceffect.

ii) Effect on compounds on Mitochondrial DNA Synthesis: a real-time PCRassay to accurately quantify mitochondrial DNA content has beendeveloped (see Stuyver L J, Lostia S, Adams M, Mathew J S, Pai B S,Grier J, Tharnish P M, Choi Y, Chong Y, Choo H, Chu C K, Otto M J,Schinazi R F. Antiviral activities and cellular toxicities of modified2′,3′-dideoxy-2′,3′-didehydrocytidine analogs. Antimicrob. AgentsChemother. 2002; 46: 3854-60). This assay was used in all studiesdescribed in this application that determine the effect of test compoundon mitochondrial DNA content. In this assay, low-passage-number HepG2cells were seeded at 5,000 cells/well in collagen-coated 96-well plates.Compounds were added to the medium to obtain final concentrations of 0μM, 0.1 μM, 10 μM and 100 μM. On culture day 7, cellular nucleic acidswere prepared by using commercially available columns (RNeasy 96 kit;Qiagen). These kits co-purify RNA and DNA, and hence, total nucleicacids were eluted from the columns. The mitochondrial cytochrome coxidase subunit II (COXII) gene and the β-actin or rRNA gene wereamplified from 5 μl of the eluted nucleic acids using a multiplex Q-PCRprotocol with suitable primers and probes for both target and referenceamplifications. For COXII the following sense, probe and antisenseprimers are used, respectively:5′-TGCCCGCCATCATCCTA-3′,5′-tetrachloro-6-carboxyfluorescein-TCCTCATCGCCCTCCCATCCC-TAMRA-3’and 5′-CGTCTGTTATGTAAAGGATGCGT-3′. For exon 3 of the β-actin gene(GenBank accession number E01094) the sense, probe, and antisenseprimers are 5′-GCGCGGCTACAGCTTCA-3′,5′-6-FAMCACCACGGCCGAGCGGGATAMRA-3′and 5′-TCTCCTTAATGTCACGCACGAT-3′, respectively. The primers and probesfor the rRNA gene are commercially available from Applied Biosystems.Since equal amplification efficiencies were obtained for all genes, thecomparative CT method was used to investigate potential inhibition ofmitochondrial DNA synthesis. The comparative CT method uses arithmeticformulas in which the amount of target (COXII gene) is normalized to theamount of an endogenous reference (the β-actin or rRNA gene) and isrelative to a calibrator (a control with no drug at day 7). Thearithmetic formula for this approach is given by 2-ΔΔCT, where ΔΔCT is(CT for average target test sample—CT for target control)—(CT foraverage reference test—CT for reference control) (see Johnson M R, KWang, J B Smith, M J Heslin, R B Diasio. Quantitation ofdihydropyrimidine dehydrogenase expression by real-time reversetranscription polymerase chain reaction. Anal. Biochem. 2000;278:175-184). A decrease in mitochondrial DNA content in cells grown inthe presence of drug would indicate mitochondrial toxicity.

iii) Electron Microscopic Morphologic Evaluation: NRTI induced toxicityhas been shown to cause morphological changes in mitochondria (e.g.,loss of cristae, matrix dissolution and swelling, and lipid dropletformation) that can be observed with ultrastructural analysis usingtransmission electron microscopy (see Cui L, Schinazi R F, Gosselin G,Imbach J L. Chu C K, Rando R F, Revankar G R, Sommadossi J P. Effect ofenantiomeric and racemic nucleoside analogs on mitochondrial functionsin HepG2 cells. Biochem. Pharmacol. 1996, 52, 1577-1584; Lewis W, LevineE S, Griniuviene B, Tankersley K O, Colacino J M, Sommadossi J P,Watanabe K A, Perrino F W. Fialuridine and its metabolites inhibit DNApolymerase gamma at sites of multiple adjacent analog incorporation,decrease mtDNA abundance, and cause mitochondrial structural defects incultured hepatoblasts. Proc Natl Acad Sci USA. 1996; 93: 3592-7;Pan-Zhou X R, L Cui, X J Zhou, J P Sommadossi, V M Darley-Usmar.Differential effects of antiretroviral nucleoside analogs onmitochondrial function in HepG2 cells. Antimicrob. Agents Chemother.2000, 44, 496-503). For example, electron micrographs of HepG2 cellsincubated with 10 μM fialuridine (FIAU; 1,2′-deoxy-2′-fluoro- 1-D-arabinofuranosly-5-iodo-uracil) showed the presence of enlargedmitochondria with morphological changes consistent with mitochondrialdysfunction. To determine if compounds promoted morphological changes inmitochondria, HepG2 cells (2.5×10⁴ cells/mL) were seeded into tissuecultures dishes (35 by 10 mm) in the presence of 0 ρM, 0.1 ρM, 1 μM, 10μM and 100 μM test compound. At day 8, the cells were fixed, dehydrated,and embedded in Eponas described previously. Thin sections wereprepared, stained with uranyl acetate and lead citrate, and thenexamined using transmission electron microscopy.

Example 4

Assay for Bone Marrow Cytotoxicity

Primary human bone marrow mononuclear cells were obtained commerciallyfrom Cambrex Bioscience (Walkersville, Md.). CFU-GM assays were carriedout using a bilayer soft agar in the presence of 50 units/mL humanrecombinant granulocyte/macrophage colony-stimulating factor, whileBFU-E assays used a methylcellulose matrix containing 1 unit/mLerythropoietin (see Sommadossi J P, Carlisle R. Toxicity of3′-azido-3′-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl)guaninefor normal human hepatopoietic progenitor cells in vitro. Antimicrob.Agents Chemother. 1987; 31: 452-454; Sommadossi, J P, Schinazi, R F,Chu, C K, and Xie, M Y. Comparison of Cytotoxicity of the (−) and (+)enantiomer of 2′,3′-dideoxy-3′-thiacytidine in normal human bone marrowprogenitor cells. Biochem. Pharmacol. 1992; 44:1921-1925). Eachexperiment was performed in duplicate in cells from three differentdonors. AZT was used as a positive control. Cells were incubated in thepresence of the compound for 14-18 days at 37° C. with 5% CO₂, andcolonies of greater than 50 cells are counted using an invertedmicroscope to determine IC₅₀. The 50% inhibitory concentration (IC₅₀)was obtained by least-squares linear regression analysis of thelogarithm of drug concentration versus BFU-E survival fractions.Statistical analysis was performed with Student's t test for independentnon-paired samples.

Example 5

Cytotoxicity Assay

The toxicity of the compounds was assessed in Vero, human PBM, CEM(human lymphoblastoid), MT-2, and HepG2 cells, as described previously(see Schinazi R. F., Sommadossi J.-P., Saalmann V., Cannon D. L., XieM.-Y., Hart G. C., Smith G. A. & Hahn E. F. Antimicrob. AgentsChemother. 1990, 34, 1061-67). Cycloheximide was included as positivecytotoxic control, and untreated cells exposed to solvent were includedas negative controls. The cytotoxicity IC₅₀ was obtained from theconcentration-response curve using the median effective method describedpreviously (see Chou T.-C. & Talalay P. Adv. Enzyme Regul. 1984, 22,27-55; Belen'kii M. S. & Schinazi R. F. Antiviral Res. 1994, 25,1-11).

The data for 10 is

-   PBM>100 μM (17% inhibition@100 μM)-   CEM>100 μM (11% inhibition@100 μM)-   VERO>100 (−0.8% inhibition@100 μM)

Example 6

HCV Replicon Assay¹

Huh 7 Clone B cells containing HCV replicon RNA would be seeded in a96-well plate at 5000 cells/well, and the compounds tested at 10 μM intriplicate immediately after seeding. Following five days incubation(37° C., 5% CO₂), total cellular RNA was isolated by using versaGene RNApurification kit from Gentra. Replicon RNA and an internal control(TaqMan rRNA control reagents, Applied Biosystems) were amplified in asingle step multiplex Real Time RT-PCR Assay. The antiviraleffectiveness of the compounds was calculated by subtracting thethreshold RT-PCR cycle of the test compound from the threshold RT-PCRcycle of the no-drug control (ΔCt HCV). A ΔCt of 3.3 equals a 1-logreduction (equal to 90% less starting material) in Replicon RNA levels.The cytotoxicity of the compounds was also calculated by using the ΔCtrRNA values. (2′-Me-C) was used as the control. To determine EC₉₀ andIC₅₀ values², ΔCt: values were first converted into fraction of startingmaterial³ and then were used to calculate the % inhibition.

REFERENCES

-   1. Stuyver L et al., Ribonucleoside analogue that blocks replication    or bovine viral diarrhea and hepatitis C viruses in culture.    Antimicrob. Agents Chemother. 2003, 47, 244-254.-   2. Reed I J & Muench H, A simple method or estimating fifty percent    endpoints. Am. J. Hyg. 27: 497, 1938.-   3. Applied Biosystems Handbook

The results are shown in Table 1 below:

TABLE 1 Compound Conc (μM) DCt HCV DCt rRNA HCV rRNA EC50 EC90 CC50 100.01 1.16 −0.21 54.99 −15.99 0.01 0.03 >0.3 0.03 3.29 −0.49 89.67 −40.100.1 6.05 −0.09 98.47 −6.27 0.3 8.72 0.14 99.76 9.35

Example 7

Bioavailability Assay in Cynomolgus Monkeys

The following procedure can be used to determine whether the compoundsare bioavailable. Within 1 week prior to the study initiation, acynomolgus monkey can be surgically implanted with a chronic venouscatheter and subcutaneous venous access port (VAP) to facilitate bloodcollection and can undergo a physical examination including hematologyand serum chemistry evaluations and the body weight recording. Eachmonkey (six total) receives compound at a dose level of 2-20 mg/kg,either via an intravenous bolus (3 monkeys, IV), or via oral gavage (3monkeys, PO). Each dosing syringe is weighed before dosing togravimetrically determine the quantity of formulation administered.Urine samples are collected via pan catch at the designated intervals(approximately 18-0 hours pre-dose, 0-4, 4-8 and 8-12 hours post-dosage)and processed. Blood samples are collected as well (pre-dose, 0.25, 0.5,1,2, 3,6, 8, 12 and 24 hours post-dosage) via the chronic venouscatheter and VAP or from a peripheral vessel if the chronic venouscatheter procedure should not be possible. The blood and urine samplesare analyzed for the maximum concentration (Cmax), time when the maximumconcentration is achieved (Tmax), area under the curve (AUC), half-lifeof the dosage concentration (TV,), clearance (CL), steady state volumeand distribution (Vss) and bioavailability (F).

Example 8

Effect of HCV Protease Inhibitors on Selected Human Proteases

HCV protease inhibitors have demonstrated great antiviral potency inaddition to interesting toxicities associated with inhibition of hostproteases. In an effort to circumvent similar toxicities, new proteaseinhibitors were evaluated for inhibition of a panel of important humanproteases. The enzymes tested are Elastase (Neutrophil), Plasmin,Thrombin, and Cathepsin S.

Neutrophil elastase (or leukocyte elastase) also known as ELA2 (elastase2) is a serine protease in the same family as chymotrypsin and has broadsubstrate specificity. Secreted by neutrophils during inflammation, oneof its primary roles is to destroy bacteria in host tissue. (Belaaouajet al, Science 289 (5482): 1185-8).

Plasmin is a serine protease derived from the conversion of plasminogenin blood plasma by plasminogen activators (Collen, D. Circulation, 93,857 (1996). This enzyme (EC 3.4.21.7) degrades many blood plasmaproteins, most notably, fibrin clots. Plasmin is also involved inseveral pathological and physiological processes such as inflammation,neoplasia, metastasis, wound healing, angiogenesis, embryogenesis andovulation (Vassalli, J. D. et al, J. Clin. Invest. 88, 1067 (1991).

Thrombin is a coagulation protein in the blood stream that has manyeffects in the coagulation cascade, the last enzyme in the clottingcascade. It is a serine protease that converts soluble fibrinogen intoinsoluble strands of fibrin, as well as catalyzing many othercoagulation-related reactions.

Cathepsin S, a member of the peptidase C1 family, is a lysosomalcysteine protease that may participate in the degradation of antigenicproteins to peptides for presentation on MHC class II molecules,therefore it is key to immune response. The encoded protein can functionas an elastase over a broad pH range.

Materials:

-   -   Victor 3 Plate reader (Perkin Elmer)    -   Clear 96 well Plates (Phenix Research)    -   Black 96 well Plates (Perkin Elmer)    -   RNase and Dnase pure water

Methods:

Elastase (Human Neutrophil (Cat #16-14-051200 Athens Research andTechnology, Athens Ga.)): Reactions were conducted in a sample volume of100 μL per well in a clear 96 well plate. A 2× assay buffer was madecontaining 200 mM Tris-HCl (pH 7.5), 150 mM NaCl and 50% glycerol. Foreach sample 50 μL 2× assay buffer was added to each well. The substrate(MeOSuc-AAPV-pNA, Chromogenic Substrate, Cat #P-213, Enzo Life Sciences,Plymouth Meeting, Pa.; 50 mM stock in DMSO) was added to a finalconcentration of 1 mM. The drug dilutions were added (25 μL) at 4×concentrations in water. Finally, a mixture was made of 1 μL elastaseand 22 μL water for each sample and 23 μL was added to each well. Thesamples were incubated at room temperature for 30 min. The absorbance at405 nM was read on the Victor 3 plate reader. All samples were tested induplicate. Results are shown as blank adjusted (no Elastase) percentagesof maximum absorbance, which was given by a no inhibitor control.

Plasmin and Thrombin(Sensolyte RH110 Plasmin Activity Assay Kit andSensolyte Thrombin Activity Assay Kit (Anaspec)): Reactions wereconducted in a sample volume of 100 μL per well in a black 96 wellplate. Protocol A was followed from the kit insert where the 2× assaybuffer was diluted 1:1 with deionized water. Included in each assay werea positive control (diluted enzyme and no test compound), inhibitorcontrol (contains diluted enzyme and plasmin inhibitor; component E fromthe kit or thrombin inhibitor; N-α-NAPAP synthetic inhibitor) andsubstrate control (assay buffer and substrate). Vehicle andautofluorescence controls were also performed. Drug dilutions were added(10 μL) at 10× concentrations in assay buffer. The enzyme was added at40 μL/well at a concentration of 0.25 μg/mL (plasmin) and 1 μg/mL(thrombin) to all wells except the substrate control. Finally, 50 μLassay buffer containing substrate was added to each well. The substratewas added to a final concentration of 50 nM (plasmin) or 20 nM(Thrombin). The samples were incubated at room temperature for 30 min.The fluorescence intensity was read on the Victor 3 plate reader atEx/Em=490 nm/520 nm. All samples were tested in duplicate. Results areshown as substrate control adjusted percentages of maximum absorbance,which was given by the positive control.

Cathespsin S (Sensolyte Cathepsin S Activity Assay Kit (Anaspec)):Reactions were conducted in a sample volume of 100 μL per well in ablack 96 well plate. Protocol A was followed from the kit insert whereDTT was added to assay buffer to yield a 5 μM concentration. Included ineach assay were a positive control (diluted enzyme and no testcompound), inhibitor control (contains diluted enzyme and plasmininhibitor; component E or thrombin inhibitor; N-a-NAPAP syntheticinhibitor) and substrate control (assay buffer and substrate). Vehicleand autofluorescence controls were also performed. Drug dilutions wereadded (10 μL) at 10× concentrations in assay buffer. The cathepsin S wasadded at 40 μL/well at a concentration of 2.5 μg/mL to all wells exceptthe substrate control. Finally, 50 82 L of assay buffer containingsubstrate was added to each well. The substrate was added to a finalconcentration of 16 nM. The samples were incubated at room temperaturefor 30 min. The fluorescence intensity was read on the Victor 3 platereader at Ex/Em=490 nm/520 nm. All samples were tested in duplicate.Results are shown as substrate control adjusted percentages of maximumabsorbance, which was given by the positive control.

The results are shown in Table 2, below:

TABLE 2 Inhibition of Human Proteases (IC₅₀, μM) Drug Elastase CathepsinG Chymo-trypsin Kallikrein Plasmin Thrombin Cathepsin S Chymase10 >100 >100 >100 47 >100 >100 >100 >100

Example 9 Activity of Compounds Versus Hepatitis C Virus NS3/4A WT andMutant Protease

The HCV NS3/4A protease assays were carried out using a SensoLyte® 490HCV Protease Assay Kit using fluorescence resonance energy transfer(FRET) peptide (AnaSpec).

The results are shown in Tables 3 and 4 below:

TABLE 3 Anti-HCV protease activity Wild Type A156T R155K D168V V36M CodeEC₅₀, μM EC₉₀, μM EC50, μM EC90, μM EC50, μM EC90, μM EC50, μM EC90, μMEC50, μM EC90, μM 10 0.0049 0.030 0.033 0.130 0.27 2.7 0.040 0.6300.0034 0.0455 stdev 0.0017 0.029 0.030 NA 0.17 3.0 0.043 NA 0.00370.0488 Fold Increase 1.0 1.0 6.7 4.4 55.8 91.4 8.1 21.1 0.7 1.5

TABLE 4 Anti-HCV protease activity A156S V170A D168A T54A Code EC50, μMEC90, μM EC50, μM EC90, μM EC50, μM EC90, μM EC50, μM EC90, μM 10 0.0080.085 0.0055 0.0095 0.57 8.30 0.07 0.85 stdev 0.000 0.021 0.0007 0.00070.01 NA 0.03 NA Fold Increase 1.6 2.9 1.1 0.3 115.3 278.5 15.0 28.5

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

1. A compound of Formula (I):

or a pharmaceutically acceptable salt, prodrug, or solvate thereof,wherein J and J¹ can be present or absent, and when present, areindependently selected from lower alkyl (C₁-C₆), aryl, arylalkyl,alkoxy, aryloxy, heterocyclyl, heterocyclyloxy, keto, hydroxy, amino,arylamino, carboxyalkyl, carboxamidoalkyl, halo, cyano, formyl,sulfonyl, or sulfonamido; and R is alkyl (C₁-C₁₀), cycloalkyl (C₃-C₈),aryl, heteroaryl, or heterocyclyl.
 2. The compound of claim 1, whereinthe compound, or pharmaceutically acceptable salt, prodrug, or solvatethereof, is in the form of individual enantiomers, stereoisomers,rotamers, tautomers, racemates, or mixtures thereof.
 3. A method fortreating or preventing an HCV infection, or reducing the biologicalactivity of HCV, comprising administering a therapeutically orprophylactically effective amount of a compound of claim 1 to a patientin need of treatment or prophylaxis thereof.
 4. The method of claim 3,wherein the compound is administered in combination with anotheranti-HCV agent.
 5. A method for inhibiting serine protease activity,comprising the step of administering to said patient a compoundaccording to claim
 1. 6. The method of claim 3, wherein said compound isadministered to a patient and is formulated together with apharmaceutically suitable carrier into a pharmaceutically acceptablecomposition.
 7. The method of claim 4, wherein said compound isadministered to a patient and is formulated together with apharmaceutically suitable carrier into a pharmaceutically acceptablecomposition.
 8. The method of claim 5, wherein said compound isadministered to a patient and is formulated together with apharmaceutically suitable carrier into a pharmaceutically acceptablecomposition.